Organ transplantation, the most effective therapy for organ failure, is currently limited by a severe lack of available donor organs. Tissue engineering is a promising solution to organ shortage. A barrier to construction of complex replacement organs is creating a functional microvascular system within the tissue engineered graft to provide adequate perfusion. Human endothelial cells (ECs) can self-assemble into microvascular conduits when implanted into immunodeficient mice, but evolution into a fully functional microvascular system involves vessel remodeling and recruitment of mural cells, particularly pericytes (PCs), control of paracellular leak through formation of inter-endothelial tight junctions (TJs), and appropriate anastomoses between different segments of the organ. To benefit patients with organ failure, grafts will likely have to be constructed in advance from allogeneic cells. But human ECs, which are required for graft perfusion, initiate allograft rejection. We will test several strategies to address these unsolved issues, using innovative approaches that alter the graft microenvironment or that alter the ECs through genetic engineering, exploiting a method we developed for CRISPR/Cas9 modification of ECs derived from umbilical cord blood progenitor cells (HCBECs). We will test our approaches using in vitro and in vivo models of microvessel formation either by self-assembly single EC suspensions or by sprouting from EC spheroids. In aim 1a, we will optimize vessel complexity and PC investment of EC tubes, initially using Bcl-2 transduction. We will also examine the effects of VEGF-A delivery from alginate microparticles or sensitizing HCBECs to VEGF-A by altering Ras signaling to increase EC tube formation, or increasing PC investment by enhancing EC production of PDGF-BB. In specific aim 1b, we will determine if TJ formation by HCBECs can be increased by sustained release of a Tie2 activating drug or by a cAMP-inducing agent to target the cAMP/Epac1/Rap1/Rac-1 pathways of barrier strengthening. Alternatively, we will modify HCBECs to enhance their sensitivity to Tie 2 agonists or to mimic the response to cAMP. In aim 1c, we will optimize perfusion of co-engrafted rat glomeruli by human HCBEC-derived microvessels by manipulating the balance of VEGF-A, semaphorin 3a and semaphorin 3c signals, or by altering the responses of HCBECs to these agents through changes in neuropilin expression. In aim 2a, we will alter the gel microenvironment to create a zone of immune-privilege through sustained release of rapamycin, an immunosuppressive drug, or of IL-10, an immunosuppressive cytokine, or by co-engraftment of encapsulated smooth muscle cells that physiologically create a site of immune-privilege in the vessel wall. In Aim 2b, we will geneticall alter HCBECs to remove signals necessary for T cell activation, namely MHC molecules and CD58, or overexpress inhibitory signals, namely PD-L1 and PD-L2. Successful outcomes of these investigations will identify approaches that may be broadly applicable to tissue engineering.